Antibodies are large glycoproteins, secreted by B-lymphocyte derived plasma cells in response to an antigen. There are five major classes of antibodies: IgG, IgM, IgA, IgE, and IgD. IgG represents about 75% of serum antibodies in human body and it is the most common type of antibody found in the circulation. In all the five major classes of antibodies the basic unit is a Y-shaped monomer, which consists of two identical heavy chains (HC) and two identical light chains (LC). Each LC has one variable domain (VL) and one constant domain (CL). Each HC has one variable domain (VH) and three constant domains (CH). The ‘arms’ in the Y-structure form the antigen binding fragment (Fab). The arms are connected by a flexible hinge region to homodimeric Fc fragment (Fragment crystallizable) which forms the ‘base’ of the Y structure. The ability of an antibody to communicate with the other components of the immune system is mediated via the Fc-region. Production of homodimeric Fc-regions in mammalian cell lines is known in the art. Such homodimeric Fc regions may be used for example to bring antibody like qualities to fusion proteins.
A bispecific antibody (BsAb) is an artificial protein that is composed of fragments of two different antibodies and consequently BsAb binds to two different types of antigens. Bispecific antibodies belong to multispecific antibodies. Multispecific antibodies may be bispecific, trispecific, or quardo-specific antibodies. The Fc-fragments of such multispecific antibodies are preferably heterodimers. Bispecific, as well as other multispecific antibodies in general have shown tremendous potential in a broad range of clinical and diagnostic applications. There are two bispecific antibody drugs approved in European Union and in the US for treatment of oncological diseases (Catumaxomab and Blinatumab). Due to their unique features, bispecific and multispecific antibodies generally have staged to be a very attractive format for next generation of antibody therapeutics.
In the clinical research area, diagnostic applications for specific antibodies have been described in several publications (e.g. Fanger et al. Crit. Rev. Immunol. 1992, 12:101-124; Nolan, et al. Biochem. Biophys. Acta. 1990, 1040:1-11; Song-Sivilai et al. Clin Exp. Immunol. 1990, 79:315). However, the most impressive application for bispecific antibodies (BsAb) reside in immune-oncology field. Theoretically, one arm of BsAb can bind to a tumor antigen on the tumor cells, and the second arm of the BsAb can bind to an immune effector cell marker. Thus, BsAbs can serve as a bridging agent to recruit immune effector cells (natural killer cells or effector T-cells) and bring them to the local tumor site to kill tumor cells. Those therapeutic applications have been described in numerous publications (Hseih-Ma et al., Cancer Res. 1992, 52:6832-6839; Weiner et al., Cancer Res. 1993, 53: 94-100; Shalaby et al. J. Exp. Med. 1992, 175: 217; Weiner et al., J. Immunol. 1994, 152:2385).
Several different ways to make BsAbs or multispecific antibodies are known in the art. In the 1980's, bispecific antibodies were generated by cross-hybrid of two different hybridomas (Millstein and Cello, Nature 1983, 305: 537-539). Because of the random pairing of two different heavy chains and two different light chains, those hybrid hybridomas (also called quadromas) could generate up to 10 different kinds of antibody combinations, only one of which was the desired BsAb-format. This situation resulted in cumbersome and low yield purification of the correct BsAb-format. To overcome the random assortment problems, it is preferred to create two differently modified Fc-domains such that those two modified Fc domains are able to favor heterodimerc formation over homodimeric formation when they meet each other. Each modified Fc can be fused with a Fab or ScFv with a unique binding specificity. When those two differently modified Fc containing fragments with different binding specificities are co-expressed in a mammalian cell culture, they can form a heterodimeric bispecific antibody with a favorable yield.
Another approach is disclosed in PCT patent application publication WO2007/147901 and in U.S. Pat. No. 8,592,562, in which the electric charges of the first CH3 domain and the second CH3 domain are re-distributed such that those two differently modified CH3 domains will favor heterodimer formation over homodimer formation. However, generally, co-expressing two different Fc heavy chains in mammalian cells may result to formation of some heterodimeric Fc fragments, but also to substantial formation of homodimeric fragments. Purification of the heterodimeric fragments from the co-transfected cell culture or engineered stable cell line supernatant is cumbersome and expensive.
Certain improvements have been introduced to this problem. U.S. Pat. No. 5,807,706 for example, discloses so called ‘knob-into-hole’ mutations at the CH3 domain. By employing the ‘knob into hole” strategy, several other methods have been disclosed for example in US patent applications 2014/0348839 and 2013/0245233). This technology resulted in higher formation of heterodimers, but some ‘hole’ homodimers and ‘knob’ monomers were still present. US patent application number 2012/0116057 disclosed an improvement in heterodimerization by substituting serine at position of 364 preferably with alanine in the wild type CH3 domain. Such substitution led to increased aggregation of heterodimeric Fc.
Antibodies have become increasingly important in developing therapeutic compositions. Multispecific antibody derivatives, including bispecific antibodies may be considered as the next generation of targeted biologics for cancer therapy. Multispecific antibodies bind at least two antigens or epitopes. Application of multispecific antibodies in experimental cancer therapy includes molecules that bind different cell surface proteins to achieve more complete blockage of proliferative or angiogenesis-associated pathways. Multispecific antibodies can also be applied as vehicles to deliver immune effector cells to tumors. Accordingly, multispecific antibodies are desirable due to their capability to bind more than one antigen. However, making and purifying multispecific antibodies, including bispecific antibodies, is still technical challenge and also very expensive. Therefore, new methods are required to make such antibodies with high purity.
Various implements are known in the art, but fail to address all of the problems solved by the invention described herein. One embodiment of this invention is illustrated in the accompanying drawings and will be described in more detail herein below.